Methods of purifying antibodies

ABSTRACT

The invention provides methods of purifying antibodies using various antibody-specific purification media to rapidly and efficiently separate mixtures of antibodies, antibody fragments and/or antibody components to isolate a desired antibody product from the mixture. The invention relates to the purification of bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, e.g., antibodies composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other a Lambda constant domain, including antibodies of different specificities that share a common heavy chain. The invention also provides the methods of efficiently purifying intact antibodies by separating the intact antibody from non-intact antibodies including free light chains.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/655,955, filed Oct. 19, 2012, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 61/548,958, filedOct. 19, 2011, which is herein incorporated by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named NOVI-026_C01US_SeqListing which wascreated on Jul. 11, 2018 and is 10.0 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The invention provides methods of purifying antibodies using variousantibody-specific affinity media to rapidly and efficiently separatemixtures of antibodies, antibody fragments and/or antibody components toisolate a desired antibody product from the mixture. The inventionrelates to the purification of bispecific monoclonal antibodies carryinga different specificity for each binding site of the immunoglobulinmolecule, e.g., antibodies composed of a single heavy chain and twodifferent light chains, one containing a Kappa constant domain and theother a Lambda constant domain, including antibodies of differentspecificities that share a common heavy chain. The invention alsoprovides the methods of efficiently purifying intact antibodies byseparating the intact antibody from free light chains produced duringthe antibody cell culture expression process.

BACKGROUND OF THE INVENTION

An antibody is composed of four polypeptides: two heavy chains and twolight chains. The antigen binding portion of an antibody is formed bythe light chain variable domain (VL) and the heavy chain variable domain(VH). At one extremity of these domains six loops form the antigenbinding site and also referred to as the complementarity determiningregions (CDR). Three CDRs are located on the VH domain (H1, H2 and H3)and the three others are on the VL domain (L1, L2 and L3).

The vast majority of immunoglobulins are bivalent and monospecificmolecules carrying the same specificity on both arms as they arecomposed of two identical heavy chain polypeptides and two identicallight chain polypeptides.

Monoclonal antibodies have emerged as a successful and attractive classof molecules for therapeutic intervention in several areas of humandisease. However, targeting or neutralizing a single protein is notalways sufficient to achieve efficacy in certain diseases which limitsthe therapeutic use of monoclonal antibodies. It is increasingly clearthat in a number of indications neutralizing one component of abiological system is not sufficient to achieve efficacy. One solution tothis problem is the co-administration of several monoclonal antibodies.This approach is however complicated by regulatory aspects if theantibodies to be combined have not been previously approvedindividually. Moreover, combination approaches are also costly from amanufacturing perspective. Accordingly, there exists a need forantibodies and therapeutics that enable targeting of multiple antigenswith a single molecule, as well as a need for efficiently purifying andisolating these multi-specific antibodies. There also exists a need forefficiently purifying and isolating intact antibodies from mixtures thatcontain antibodies, antibody fragments and/or antibody components.

SUMMARY OF THE INVENTION

The invention provides a variety of techniques that useantibody-specific purification media and related reagents to separateand isolate a desired antibody product or combination of desiredantibody products from a mixture of antibodies, antibody fragments,antibody components such as free light chains, and combinations thereof.The methods provided herein rapidly and efficiently separate a desiredantibody product or combination of desired antibody products from amixture of antibodies and/or fragments thereof. For example, in someembodiments, the methods are designed to isolate an intact antibody or acombination of intact antibodies from antibody components such as freelight chains, which are a by-product of the antibody manufacturingprocess. As used herein, the term “intact” antibody molecule means afull-length antibody, as opposed to a fragment and/or other portion of afull-length antibody, which are referred to herein collectively as a“non-intact” antibody. The intact antibody can be any intact antibody,including by way of non-limiting example, intact monovalent antibodies,intact bispecific antibodies, intact multi-specific antibodies, intactmonoclonal antibodies, such as intact fully human antibodies, intacthumanized antibodies and/or other intact chimeric antibodies. In someembodiments, the methods are designed to isolate a bispecific antibody,such as, for example, bispecific antibodies that have a single heavychain and at least one kappa (κ) light chain region (or light chainregion derived from a κ light chain) and at least one lambda (λ) lightchain region (or a light chain region derived from a λ light chain).

The purification medium is, in some embodiments, an affinity medium, forexample, a resin or other separation means that is specific for κ lightchains and portions thereof, such as KappaSelect resin and/or a ProteinL-containing resin, which isolate antibodies and fragments thereof thatcontain a κ light chain (or a portion thereof). The purification mediumis, in some embodiments, an affinity medium, for example, a resin orother separation means that is specific for λ light chains and portionsthereof, such as LambdaFabSelect resin, which isolates antibodies andfragments thereof that contain a λ light chain (or a portion thereof).The purification medium is, for example, a mixed mode chromatographyagent such as Mep HyperCel™ chromatography sorbent, which isolatesintact IgG antibodies from antibody fragments and other antibodycomponents, including free light chains. The purification media is, insome embodiments, a combination of one or more of these media.

In one aspect, the invention allows for the purification of bispecificantibodies and antigen-binding fragments thereof that areundistinguishable in sequence from standard antibodies. The unmodifiednature of the purified antibodies and antigen-binding fragments thereofprovides them with favorable manufacturing characteristics similar tostandard monoclonal antibodies.

The methods provided herein are useful for purifying a variety ofbispecific antibodies and antigen-binding fragments thereof,particularly the bispecific antibodies referred to herein as “κλ-bodies”and antigen-binding fragments thereof, which have a common IgG heavychain and two different light chains, one having a kappa (κ) constantregion and the other having a lambda (λ) constant region, that drivespecificity for two independent targets.

The bispecific antibodies and antigen-binding fragments thereof to bepurified can be generated using any of a variety of methods. Forexample, the bispecific antibodies and antigen-binding fragments thereofcan be generated by (i) isolating two antibodies having differentspecificities and sharing the same variable heavy chain domain butdifferent variable light chains, for example by using antibody librarieshaving a fixed heavy chain or transgenic animals containing a single VHgene; (ii) fusing the variable heavy chain domain to the constant regionof a heavy chain, fusing one light chain variable domain to a Kappaconstant domain, and fusing the other variable light chain domain to aLambda constant domain; and (iii) co-expressing the three chains in ahost cell or cell line, for example, mammalian cells and/or mammaliancell lines, leading to the assembly and secretion in the supernatant ofa mixture of three antibodies: two monospecific antibodies and onebispecific antibody carrying two different light chains. In someantibodies and antigen-binding fragments thereof produced using thismethod, at least a first portion of the first light chain is of theKappa type and at least a portion of the second light chain is of theLambda type. In some antibodies and antigen-binding fragments thereofproduced using this method, the first light chain includes at least aKappa constant region. In some antibodies and antigen-binding fragmentsthereof produced using this method, the first light chain furtherincludes a Kappa variable region. In some antibodies and antigen-bindingfragments thereof produced using this method, the first light chainfurther includes a Lambda variable region. In some antibodies andantigen-binding fragments thereof produced using this method, the secondlight chain includes at least a Lambda constant region. In someantibodies and antigen-binding fragments thereof produced using thismethod, the second light chain further includes a Lambda variableregion. In some antibodies and antigen-binding fragments thereofproduced using this method, the second light chain further includes aKappa variable region. In some antibodies and antigen-binding fragmentsthereof produced using this method, the first light chain includes aKappa constant region and a Kappa variable region, and the second lightchain includes a Lambda constant region and a Lambda variable region. Insome antibodies and antigen-binding fragments thereof produced usingthis method, the constant and variable framework region sequences arehuman.

The bispecific antibodies and antigen-binding fragments thereofgenerated using this method or any other suitable method known in theart are purified using standard chromatography techniques used forantibody purification. The bispecific antibodies and antigen-bindingfragments thereof generated using this method or any other suitablemethod known in the art can also be purified using other separationtechniques, such as by way of non-limiting and non-exhaustive example,membrane filtration techniques and protein precipitation techniques. Ina preferred embodiment, the bispecific antibody (or antibodies) andantigen-binding fragment(s) thereof is purified using affinitychromatography, for example KappaSelect affinity chromatography,LambdaFabSelect chromatography or Protein L affinity chromatography.

The invention provides methods of purifying a bispecific monoclonalantibody carrying a different specificity in each combining site andconsisting of two copies of a single heavy chain polypeptide and a firstlight chain that includes a kappa constant region and a second lightchain that includes a lambda constant region. These methods include thesteps of: (i) providing a mixed antibody composition that includes oneor more of the bispecific monoclonal antibodies carrying a differentspecificity in each combining site and consisting of two copies of asingle heavy chain polypeptide and a first light chain that includes akappa constant region and a second light chain that includes lambdaconstant region (bispecific MAb); one or more monospecific monoclonalantibodies having two lambda light chains or portions thereof (λ-MAb);and one or more monospecific monoclonal antibodies having two kappalight chains or portions thereof (κ-MAb); (ii) providing a separationmeans that has a specific affinity for a kappa light chain constantregion or a lambda light chain constant region; (iii) contacting theseparation means with the mixed antibody composition under conditionsthat allow for differential binding to the separation means by thebispecific MAb as compared to the binding to the separation means by thekappa light chain constant regions of the κ-MAb or by the lambda lightchain constant regions of the λ-MAb; and (iv) eluting antibodies fromthe separation means under conditions that allow for preferentialdetachment of the bispecific MAb as compared to the detachment of thekappa light chain constant regions of the κ-MAb or the detachment of thelambda light chain constant regions of the λ-MAb.

In some embodiments, the separation means is a resin, a membrane, amagnetic bead, a particle or a monolith. In some embodiments, theseparation means is coupled to a ligand having high specificity andaffinity for a kappa light chain constant region or a lambda light chainconstant region. In some embodiments, the separation means is aKappaSelect resin, a LambdaFabSelect resin, or a Protein L resin. Insome embodiments, the ligand is an anti-lambda monoclonal antibody or ananti-kappa monoclonal antibody.

In some embodiments, the binding and/or elution conditions include astep variation in the pH level. In some embodiments, the binding and/orelution conditions include a step variation in the inorganic saltconcentration such as sodium chloride (NaCl) concentration or theconcentration of other inorganic salts such as by way of non-limitingand non-exhaustive example, inorganic salt combinations from theHofmeister series of ions. In some embodiments, the binding and/orelution conditions include a step variation in the concentration of anamino acid in the composition, such as by way of non-limiting andnon-exhaustive example, the concentration of arginine, histidine,proline, phenylalanine, tyrosine, tryptophan and/or glycine. In someembodiments, the binding and/or elution conditions include one or moremild denaturing agents such as by way of non-limiting and non-exhaustiveexample, Polysorbate 20, Polysorbate 80, Polyethylene glycol 2000,Polyethylene glycol 8000, Triton X-100, CHAPS, NP-40, and other ionic,non-ionic and/or zwitterionic surfactants.

In some embodiments, the methods comprise the further step ofdetermining the purity and proportions of bispecific antibody, κ-MAband/or λ MAb in the eluted fraction. This step can be accomplished usingany of a variety of art-recognized techniques, such as by way ofnon-limiting and non-exhaustive example, hydrophobic interaction-highperformance liquid chromatography (HIC-HPLC), ion exchange-highperformance liquid chromatography (IEX-HPLC) or reverse phase-highperformance liquid chromatography (RP-HPLC).

The Example provided herein demonstrates the feasibility of using ahigher pH step elution to preferentially elute bispecific κλ-bodyproduct from KappaSelect affinity resin over monospecific κ-MAb whichelutes at a lower pH, as the monospecific MAb presumably has a higheraffinity to the resin owing to the presence of two κ chains in themonospecific format as opposed to a single κ chain in the κλ-body. Themethods described herein are useful in other chromatography supportswhere affinity towards the light chain is used to differentially bindthe monospecific and/or bi-specific products, such as, by way ofnon-limiting and non-exhaustive example, LambdaFabSelect, ion-exchange,hydrophobic interaction, and mixed mode resins (e.g., hydroxyapatite)and other chromatography techniques. Those of ordinary skill in the artwill readily appreciate other art-recognized techniques that would fallwithin this category. Elution strategies to separate the differentproducts should not only be limited to pH variation, but could alsoencompass, by way of non-limiting and non-exhaustive example,cation-exchange separation techniques using step variation of saltconcentration such as NaCl concentration or the concentration of otherinorganic salts (e.g., inorganic salt combinations from the Hofmeisterseries of ions), Arginine and other amino acids such as histidine,proline, phenylalanine, tyrosine, tryptophan, and glycine concentration,use of mild denaturing agents such as, for example, Polysorbate 20,Polysorbate 80, Polyethylene glycol 2000, Polyethylene glycol 8000,Triton X-100, CHAPS, NP-40, and other ionic, non-ionic and/orzwitterionic surfactants, and so on.

In another aspect, the present invention relates to the efficientremoval of free light chains from intact antibodies, includingmonospecific antibodies, bispecific antibodies and mixtures of intactantibodies. In particular, chromatography conditions have beenidentified that are applicable for isolating intact bispecific ormonospecific monoclonal antibodies or combinations of intact bispecificor monospecific monoclonal antibodies from free light chains.

The Example provided herein uses a bispecific antibody for the mixedmode affinity isolation of intact IgG molecules from a mixture thatcontains free light chains. It is to be understood that this is merelyan example, and the methods provided herein are useful in conjunctionwith any antibody manufacturing process that generates incompleteantibody components (i.e., non-intact antibodies) that can be present asmonomers or polymers, and in native or altered conformation. Inaddition, while the examples provided herein use a purification mediumthat identifies intact IgG antibodies, it is to be understood that thesemethods can be used with any purification media that identify otherintact antibody molecules.

The intact antibody molecules generated using any suitable method knownin the art can be purified using the mixed mode chromatographyseparation techniques provided herein alone or in conjunction with anyother suitable separation techniques, such as by way of non-limiting andnon-exhaustive example, membrane filtration techniques and proteinprecipitation techniques.

The invention provides methods of purifying an intact antibody or acombination of intact antibodies from a mixture that contains non-intactantibodies, including antibody components, dimers of antibodycomponents, antibody fragments and/or combinations thereof. Thecombination of intact antibodies can include one or more different typesof intact antibodies, including antibodies that bind the same ordifferent targets. The intact antibody can be any intact antibody,including by way of non-limiting example, intact monovalent antibodies,intact bispecific antibodies, intact multi-specific antibodies, intactmonoclonal antibodies, such as intact fully human antibodies, intacthumanized antibodies and/or other intact chimeric antibodies. Thecombination of intact antibodies can include any combination of intactantibodies to the same or different targets, including by way ofnon-limiting example, combinations that include one or more of thefollowing: intact monovalent antibodies, intact bispecific antibodies,intact multi-specific antibodies, intact monoclonal antibodies, intactfully human antibodies, intact humanized antibodies and/or other intactchimeric antibodies.

In some embodiments, these methods include the steps of: (i) providing amixed antibody composition that includes one or more of the intactantibodies or a combination of intact antibodies and one or morenon-intact antibody molecules, including one or more antibody componentssuch as a free light chain, one or more dimers of an antibody componentsuch as a free light chain, and/or one or more antibody fragments; (ii)providing a separation means that has a differential affinity between anintact antibody molecule as compared to the non-intact antibodymolecule; (iii) contacting the separation means with the mixed antibodycomposition under conditions that allow for differential binding to theseparation means by the intact antibody molecule or combination ofintact antibodies as compared to the binding to the separation means bynon-intact antibody molecule (e.g., one or more antibody components suchas a free light chain, one or more dimers of an antibody component suchas a free light chain, and/or one or more antibody fragments); and (iv)separating the intact antibody or combination of intact antibodies fromthe separation means and retaining the intact antibody or combination ofintact antibodies, thereby purifying the intact antibody or combinationof intact antibodies from the mixed antibody composition. In someembodiments, the intact antibody or combination of intact antibodies isseparated from the separation means by eluting the intact antibody orcombination of intact antibodies from the separation means underconditions that allow for preferential detachment of the intact antibodyor combination of intact antibodies as compared to the detachment of thenon-intact antibody molecule (e.g., one or more antibody components suchas a free light chain, one or more dimers of an antibody component suchas a free light chain, and/or one or more antibody fragments). In someembodiments, the separation means is contacted by the antibody mixturecomposition under conditions that allow for binding by the intactantibody or combination of intact antibodies to the separation means,but do not allow for binding between the non-intact antibody moleculeand the separation means. In some embodiments, the intact antibody orcombination of intact antibodies is separated from the non-intactantibody fraction by removing any unbound, non-intact antibodies fromthe mixed antibody composition or by allowing the unbound, non-intactantibodies to flow through the separation means and discarding orotherwise removing the unbound fraction. In some embodiments, thenon-intact antibody molecule is a free light chain.

In some embodiments, the separation means is a resin, a membrane, amagnetic bead, a particle or a monolith. In some embodiments, theseparation means is coupled to a ligand having differential specificityand affinity for an intact antibody molecule as compared to a non-intactantibody molecule. In some embodiments, the separation means is a mixedmode chromatography medium, such as, for example, hydrophobicinteraction-high performance liquid chromatography (HIC-HPLC) or ionexchange-high performance liquid chromatography (IEX-HPLC). In someembodiments, the mixed mode chromatography medium is Mep HyperCel™.Other mixed mode chromatography media include, for example, Capto™ MMC,Capto™ adhere, HEA HyperCel™, PPA HyperCel™, CHT™ ceramichydroxyapatite, and Nuvia™ cPrime™. In some embodiments, the ligand isan anti-antibody monoclonal antibody such as, for example, an anti-IgGantibody.

In some embodiments, the methods comprise the further step ofdetermining the purity and proportions of the intact antibody in theeluted fraction. This step can be accomplished using any of a variety ofart-recognized techniques, such as by way of non-limiting andnon-exhaustive example, size exclusion-high performance liquidchromatography (SEC-HPLC), hydrophobic interaction-high performanceliquid chromatography (HIC-HPLC), ion exchange-high performance liquidchromatography (IEX-HPLC), or reverse phase-high performance liquidchromatography (RP-HPLC).

In some embodiments, these methods include the steps of: (i) providing amixed antibody composition that includes one or more of the intactantibodies or a combination of intact antibodies and one or morenon-intact antibody molecules, including one or more antibody componentssuch as a free light chain, one or more dimers of an antibody componentsuch as a free light chain, and/or one or more antibody fragments; (ii)providing a separation means that has a differential affinity between anon-intact antibody molecule as compared to an intact antibody molecule;(iii) contacting the separation means with the mixed antibodycomposition under conditions that allow for binding to the separationmeans by the non-intact antibody molecule but do not allow for bindingto the separation means by the intact antibody or combination of intactantibodies such that the intact antibody or combination of intactantibodies remain in an unbound fraction; and (iv) retaining the unboundfraction that includes the intact antibody or combination of intactantibodies. In some embodiments, the methods may include the additionalstep of separating the non-intact antibody molecule from the separationmeans. In some embodiments, the non-intact antibody molecule is a freelight chain.

In some embodiments, the separation means is a resin, a membrane, amagnetic bead, a particle or a monolith. In some embodiments, theseparation means is coupled to a ligand having differential specificityand affinity for an intact antibody molecule. In some embodiments, theseparation means is a mixed mode chromatography medium, such as, forexample, hydrophobic interaction-high performance liquid chromatography(HIC-HPLC) or ion exchange-high performance liquid chromatography(IEX-HPLC). In some embodiments, the mixed mode chromatography medium isMep HyperCel™. Other mixed mode chromatography media include, forexample, Capto™ MMC, Capto™ adhere, HEA HyperCel™, PPA HyperCel™, CHT™ceramic hydroxyapatite, and Nuvia™ cPrime™. In some embodiments, theligand is an anti-antibody monoclonal antibody such as, for example, ananti-IgG antibody.

In some embodiments, the methods comprise the further step ofdetermining the purity and proportions of the intact antibody in theeluted fraction. This step can be accomplished using any of a variety ofart-recognized techniques, such as by way of non-limiting andnon-exhaustive example, size exclusion-high performance liquidchromatography (SEC-HPLC), hydrophobic interaction-high performanceliquid chromatography (HIC-HPLC), ion exchange-high performance liquidchromatography (IEX-HPLC), or reverse phase-high performance liquidchromatography (RP-HPLC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a schematic representation of the structure of differentκλ-body bispecific antibodies composed of two copies of a unique heavychain polypeptide and two different light chain polypeptides. Thelocations and/or arrangements of the Kappa light chain and the Lambdalight chain (or portions thereof) shown in these figures are notintended to be limiting. Those of ordinary skill in the art willappreciate that the Kappa light chain and the Lambda light chain (orportions thereof) can also be arranged so as to produce the mirror-imageof the bispecific antibodies shown in FIGS. 1A-1C. Those of ordinaryskill in the art will also appreciate that the bispecific antibodiesthat are represented in a full IgG format in FIGS. 1A-1C can also begenerated using other immunoglobulin isotypes or in other immunoglobulinformats such as F(ab′)₂. FIG. 1A. Kappa variable domain fused to a Kappaconstant domain and Lambda variable domain fused to Lambda constantdomain. FIG. 1B. Kappa variable domains fused to a Kappa constant domainand a Lambda constant domain. FIG. 1C. Lambda variable domains fused toa Kappa constant domain and a Lambda constant domain.

FIG. 2 is an illustration depicting that the expression of tri-cistronicexpression vector in CHO cells gives rise to three antibody productswith a theoretical 25:50:25 ratio for the IgG products (middle panellabeled “IgGs”) and a mixture of free light chains (FLCs) and dimers ofthese FLCs (lower panel labeled “FLCs”).

FIG. 3A is a graph depicting a representative UV-trace profile ofKappaSelect affinity chromatography using step pH elution.

FIG. 3B is an illustration depicting non-reduced and reduced SDS-PAGE ofKappaSelect elution fractions.

FIG. 3C is a graph depicting IEX-HPLC analysis of KappaSelect elutionfractions.

FIG. 4A is a graph depicting a representative UV-trace profile ofLambdaFabSelect affinity chromatography using step pH elution.

FIG. 4B is an illustration depicting non-reduced and reduced SDS-PAGE ofLambdaFabSelect elution fractions.

FIG. 4C is a graph depicting HIC-HPLC analysis of LambdaFabSelectelution fractions.

FIG. 5A is a graph depicting a representative UV-trace profile of MepHyperCel™ mixed mode chromatography using step pH elution.

FIG. 5B is an illustration depicting non-reduced and reduced SDS-PAGE ofMep HyperCel™ elution fractions.

FIG. 5C is a graph depicting HIC-HPLC analysis of Mep HyperCel™ elutionfractions.

DETAILED DESCRIPTION

The invention provides a variety of techniques that useantibody-specific affinity media and related reagents to separate andisolate a desired antibody product from a mixture of antibodies,antibody fragments, antibody components such as free light chains, andcombinations thereof. The methods provided herein rapidly andefficiently separate a desired antibody product from a mixture ofantibodies and/or fragments thereof.

The present invention provides methods of purifying bispecificantibodies that are identical in structure to a human immunoglobulin.This type of molecule is composed of two copies of a unique heavy chainpolypeptide, a first light chain variable region fused to a constantKappa domain and second light chain variable region fused to a constantLambda domain. Each combining site displays a different antigenspecificity to which both the heavy and light chain contribute. Thelight chain variable regions can be of the Lambda or Kappa family andare preferably fused to a Lambda and Kappa constant domain,respectively. This is preferred in order to avoid the generation ofnon-natural polypeptide junctions. However it is also possible to obtainbispecific antibodies of the invention by fusing a Kappa light chainvariable domain to a constant Lambda domain for a first specificity andfusing a Lambda light chain variable domain to a constant Kappa domainfor the second specificity (FIG. 1). The bispecific antibodies describedherein are also referred to as IgGκλ antibodies or “κλ-bodies,” a fullyhuman bispecific IgG format. This κλ-body format allows the affinitypurification of a bispecific antibody that is indistinguishable from astandard monoclonal antibody, e.g., a standard IgG molecule, therefore,favorable as compared to previous formats.

The κλ-bodies are generated by identifying two antibody Fv regions (eachcomposed by a variable light chain and variable heavy chain domain)having different antigen specificities that share the same heavy chainvariable domain.

The present invention also provides methods of purifying intactantibodies from mixtures that contain non-intact antibody molecules,including, for example, antibody components, dimers of antibodycomponents, antibody fragments and/or combinations thereof.

The κλ-bodies and/or intact antibodies to be purified using the methodsof the invention are generated using any of a variety of methods forgenerating antibodies. Numerous methods have been described for thegeneration of monoclonal antibodies and fragments thereof (See, e.g.,Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated hereinby reference). Fully human antibodies are antibody molecules in whichthe sequence of both the light chain and the heavy chain, including theCDRs 1 and 2, arise from human genes. The CDR3 region can be of humanorigin or designed by synthetic means. Such antibodies are termed “humanantibodies” or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by using the trioma technique; the humanB-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4:72); and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies maybe utilized and may be produced by using human hybridomas (see Cote, etal., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforminghuman B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

Monoclonal antibodies are generated, e.g., by immunizing an animal witha target antigen or an immunogenic fragment, derivative or variantthereof. Alternatively, the animal is immunized with cells transfectedwith a vector containing a nucleic acid molecule encoding the targetantigen, such that the target antigen is expressed and associated withthe surface of the transfected cells. A variety of techniques arewell-known in the art for producing xenogenic non-human animals. Forexample, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which is herebyincorporated by reference in its entirety.

Alternatively, the antibodies are obtained by screening a library thatcontains antibody or antigen binding domain sequences for binding to thetarget antigen. This library is prepared, e.g., in bacteriophage asprotein or peptide fusions to a bacteriophage coat protein that isexpressed on the surface of assembled phage particles and the encodingDNA sequences contained within the phage particles (i.e., “phagedisplayed library”).

Hybridomas resulting from myeloma/B cell fusions are then screened forreactivity to the target antigen. Monoclonal antibodies are prepared,for example, using hybridoma methods, such as those described by Kohlerand Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse,hamster, or other appropriate host animal, is typically immunized withan immunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes can be immunized in vitro.

Although not strictly impossible, the serendipitous identification ofdifferent antibodies having the same heavy chain variable domain butdirected against different antigens is highly unlikely. Indeed, in mostcases the heavy chain contributes largely to the antigen binding surfaceand is also the most variable in sequence. In particular, the CDR3 onthe heavy chain is the most diverse CDR in sequence, length andstructure. Thus, two antibodies specific for different antigens willalmost invariably carry different heavy chain variable domains.

In some embodiments, the κλ-bodies and/or intact antibodies to bepurified are generated, for example, using antibody libraries in whichthe heavy chain variable domain is the same for all the library membersand thus the diversity is confined to the light chain variable domain.Such libraries are described, for example, in co-pending applicationPCT/US2010/035619, filed May 20, 2010 and published on Nov. 25, 2010 asPCT Publication No. WO 2010/135558 and co-pending applicationPCT/US2010/057780, filed Nov. 23, 2010 and published on Jul. 14, 2011 asPCT Publication No. WO 2011/084255, each of which is hereby incorporatedby reference in its entirety. However, as the light chain variabledomain is expressed in conjunction with the heavy variable domain, bothdomains can contribute to antigen binding. To further facilitate theprocess, antibody libraries containing the same heavy chain variabledomain and either a diversity of Lambda variable light chains or Kappavariable light chains can be used in parallel for in vitro selection ofantibodies against different antigens. This approach enables theidentification of two antibodies having a common heavy chain but onecarrying a Lambda light chain variable domain and the other a Kappalight chain variable domain that can be used as building blocks for thegeneration of a bispecific antibody in the full immunoglobulin format ofthe invention. The bispecific antibodies to be purified using themethods of the invention can be of different isotypes and their Fcportion can be modified in order to alter the bind properties todifferent Fc receptors and in this way modify the effectors functions ofthe antibody as well as it pharmacokinetic properties. Numerous methodsfor the modification of the Fc portion have been described and areapplicable to antibodies of the invention. (See for example Strohl, W RCurr Opin Biotechnol 2009 (6):685-91; U.S. Pat. No. 6,528,624; U.S.Patent Application Publication No. 2009/0191199 filed Jan. 9, 2009). Themethods of the invention can also be used to purify bispecificantibodies and antibody mixtures in a F(ab′)2 format that lacks the Fcportion.

Preferably, the κλ-bodies to be purified have been optimized for theco-expression of the common heavy chain and two different light chainsinto a single cell to allow for the assembly of a bispecific antibody ofthe invention. If all the polypeptides get expressed at the same leveland get assembled equally well to form an immunoglobulin molecule thenthe ratio of monospecific (same light chains) and bispecific (twodifferent light chains) should be 50%. However, it is likely thatdifferent light chains are expressed at different levels and/or do notassemble with the same efficiency. Furthermore, light chains that escapeassembly into an intact IgG molecule may be secreted into the cellculture supernatant as “free-light chains” (FLCs). Means to modulate therelative expression of the different polypeptides to compensate fortheir intrinsic expression characteristics or different propensities toassemble with the common heavy chain include, by way of non-limitingexamples, the use of promoter(s) with variable strength(s), the use ofinternal ribosome entry sites (IRES) featuring different efficiencies orother types of regulatory elements that can act at transcriptional ortranslational levels as well as acting on mRNA stability. The modulationof the expression can also be achieved by multiple sequentialtransfections of cells to increase the copy number of individual genesexpressing one or the other light chain and thus modify their relativeexpressions.

The co-expression of the heavy chain and two light chains generates amixture of three different antibodies secreted into the cell culturesupernatant: two monospecific bivalent antibodies and one bispecificbivalent antibody. The latter has to be purified from the mixture toobtain the κλ-body of interest. The purification methods describedherein greatly facilitate the purification procedure by the use ofaffinity chromatography media that specifically interact with the Kappaor Lambda light chain constant domains such as KappaSelect affinitymedium, LambdaFabSelect affinity medium, and/or the Protein L,CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices.This affinity chromatography purification approach is efficient andgenerally applicable to bispecific antibodies, including κλ-bodies. Thisis in sharp contrast with specific purification methods that have to bedeveloped and optimized for each bispecific antibody derived fromquadromas or other cell lines expressing antibody mixtures. Indeed, ifthe biochemical characteristics of the different antibodies in themixtures are similar, their separation using standard chromatographytechnique such as ion exchange chromatography can be challenging or notpossible at all.

The co-expression of the three chains led to the assembly of threedifferent antibodies: two monospecific and one bispecific antibodies.Their theoretical relative ratios should be 1:1:2 provided theexpression levels and assembly rates are similar for both light chains.The bispecific antibodies were purified using affinity chromatographyprocedures that preferentially elute the bispecific antibodies, such asthe κλ-bodies, using affinity resins.

The co-expression of the three chains also led to the generation ofexcess free light chain in the cell culture supernatant. Such free lightchains can be potentially problematic to remove in purificationprocesses omitting, for example, protein A affinity chromatography.Free-light chains could be efficiently separated from the intactantibody mix using mixed-mode chromatography as demonstrated herein.

Previous approaches to produce and purify bispecific antibody formatsaimed at forcing the production of a homogenous bispecific moleculeusing different antibody engineering approaches were done at the expenseof productivity, scalability and stability of the product. The methodsdescribed herein provide efficient and generic means to purify thebispecific antibody from a mixture containing monovalent, monoclonalantibodies and free light chains.

EXAMPLES Example 1: Purification of Bispecific Antibodies Carrying aLambda and a Kappa Light Chain

The κλ-body is a novel bi-specific IgG format that comprising a commonIgG1 heavy chain and two different light chains that drive specificityfor two independent targets. In order to allow for an efficientpurification protocol applicable to large scale industrial processes,the format requires that one light chain contains a κ constant regionwhilst the other contains a λ constant region. (See FIG. 1).

In order to produce κλ-body, the common heavy chain and two light chainsare expressed in CHO cells using a tri-cistronic expression vector. Thisvector format allows for the construction of three products:monospecific κ monoclonal antibody (MAb), bispecific κλ-body andmonospecific λ-MAb. Assuming similar expression levels and assembly withthe heavy chain between Kappa and Lambda light chains, the theoreticalproduct ratio is 25:50:25 in addition to free light chains. (See FIG.2).

Purification of this κλ-body format can be performed by sequentialbinding to KappaSelect and LambdaFabSelect affinity resins (GEHealthcare), as described for example, in co-pending U.S. applicationSer. No. 13/210,723, filed on Aug. 16, 2011. These resins are coupledwith domain ligands having high specificity and affinity for either theκ or λ constant region. However, there exists a need for improved andcost-effective purification processes that allow for large scalepurification of the κλ-bodies and other bispecific antibodies. Removalof the protein A affinity supernatant capture step is envisioned andpossible as long as a free light chains can be removed from the mixtureprior to KappaSelect and LambdaFabSelect affinity chromatography.

With the aim of streamlining the purification process, it washypothesized that the κλ-body would bind to either KappaSelect orLambdaFabSelect resins with a weaker affinity than the correspondingmonospecific κ-MAb (for KappaSelect) or monospecific λ-MAb (forLambdaFabSelect) by-product due to the fact that it contains only one ofeach light chain rather than two for the monoclonal format (either κ orλ). Furthermore, it was hypothesized that free light chains could beseparated from intact antibody using mixed mode chromatography todirectly capture recombinant protein from the supernatant. (See FIG. 2).

The studies provided herein demonstrate the successful separation ofκλ-body from monospecific kappa Ab using step pH elution during eitherKappaSelect or LambdaFabSelect affinity chromatography.

Start Material:

For KappaSelect chromatography, the clarified 25 L wave bag fermentationsupernatant of a CHO cell transfected with a κλ bispecific expressionvector (containing one γ1 heavy chain cDNA, one κ light chain cDNA andone λ light chain cDNA) was used as the starting material forpurification. For LambdaFabSelect and mixed mode chromatography, theclarified supernatant of a BIOSTAT CultiBag STR 100 L fermentation of aCHO cell transfected with a κλ bispecific expression vector (containingone γ1 heavy chain cDNA, one κ light chain cDNA and one λ light chaincDNA) was used as the starting material for purification.

KappaSelect Chromatography Step:

An anti-IFNγ/IL-6RC (i.e., IL-6RC is the complex formed between IL-6 andIL-6R) κλ-body bispecific IgG antibody was purified using KappaSelectaffinity chromatography media (GE Healthcare). The heavy and light chainamino acid sequences of the anti-IFNγ/IL-6RC κλ-body bispecific IgGantibody are shown below:

Anti-IL6RC VKappa light chain (SEQ ID NO: 4)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWLPTTPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECAnti-IFNγ VLambda light chain (SEQ ID NO: 5)NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSQSWDGNHIVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECSCommon heavy chain (SEQ ID NO: 6)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

After column loading at 10 mg/mL and a wash step with 50 mM SodiumPhosphate, 250 mM Sodium Chloride, pH 7.0 (5 column volumes), a pHstep-elution (pH 3.0 followed by pH 2.5 and pH 2.0) was performed usinga 50 mM glycine buffer adjusted to the relevant pH. The flow through(F/T) and eluted fractions were collected and analyzed by absorbancemeasurement at 280 nm (using a NanoDrop UV-Vis spectrophotometer, ThermoScientific) in order to determine product recovery, reduced andnon-reduced SDS-PAGE (using Invitrogen Novex NuPAGE 12-well 4-20%gradient gels following manufacturer's guidelines) in order to determinethe purity and composition of the samples and ion exchange-highperformance liquid chromatography (IEX-HPLC; method described below) inorder to determine the ability of the purification process to separatethe κλ-body bispecific IgG from the two monospecific antibodyby-products.

IEX-HPLC Method:

This Ion Exchange-High Performance Liquid Chromatography (IEX-HPLC)method was used to determine the proportions of monospecific andbispecific antibody in purified samples. The IEX-HPLC method allows forthe separation of protein variants according to their chargedistribution. Samples were prepared to load 50 μg onto A BioMab NP5-SScolumn (Agilent) and a linear gradient of 10 mM sodium phosphate, 500 mMNaCl, pH 6.5 (from 0% to 100% NaCl concentration) at a flow rate of 0.8mL/min was applied in order to separate the different antibody products.UV detection at 214 nm was employed to monitor sample elution. The threepopulations were identified (according to reference standards) andanalyzed according to their percentage relative area. The percentage ofeach isoform was determined by calculating the peak area of eachcomponent relative to the total peak area.

As shown by the UV trace (blue) in FIG. 3A, the three pH step elutionsapplied to the KappaSelect chromatography resin allowed for thesequential isolation of three bound fractions. Non-reduced SDS-PAGEanalysis, shown in FIG. 3B revealed the high purity of the elutedfractions containing assembled full length antibodies as anticipated.Some free light chain products (monomer and dimer forms) were alsodetected. Reduced SDS-PAGE analysis suggested that the consecutive pHelution steps lead to the differential retention of the κλ-bodyrelatively to the two monospecific antibodies, based on light chaincomposition. The eluting fraction at pH3.0 contained equivalent levelsof both light chains whereas the pH 2.5 and pH 2.0 fractions presentedminimal or no detectable levels of λ light chain. The three boundfractions were further characterized by integrating the peak areas ofthe IEX-HPLC chromatograms (FIG. 3C). The results summarized in Table 1were in accordance with the SDS-PAGE analysis, demonstrating the vastabundance of the κλ-body (70.10%) in the first eluted fraction at pH3.0. Subsequent elution steps at pH 2.5 and pH 2.0 resulted in theelution of the monospecific κ antibody. A pH step elution strategy withthe KappaSelect resin was therefore shown to effectively separatebispecific κλ-body from monospecific κ- and λ-MAb.

TABLE 1 UV peak integration of IEX-HPLC analysis of Kappa Select boundfractions % area Samples mono-k κλ-body KappaSelect pH 3.0 29.90 70.10KappaSelect pH 2.5 58.65 41.35 KappaSelect pH 2.0 89.01 10.99

This data demonstrates the feasibility of using a higher pH step elutionto preferentially elute bispecific κλ-body product from KappaSelectaffinity resin over monospecific κ-MAb which elutes at a lower pH. Thisis presumably due to a higher affinity to the resin owing to thepresence of two κ chains in the monospecific format as opposed a singleκ chain in the κλ-body.

Thus, this separation is also useful in other chromatography supportswhere affinity towards the light chain is used to differentially bindthe monospecific and/or bi-specific products, such as, by way ofnon-limiting and non-exhaustive example, LambdaFabSelect, ion-exchange,hydrophobic interaction, and mixed mode resins (e.g., hydroxyapatite).Those of ordinary skill in the art will readily appreciate otherart-recognized techniques that would fall within this category. Elutionstrategies to separate the different products should not only be limitedto pH variation, but could also encompass, by way of non-limiting andnon-exhaustive example, cation-exchange separation techniques using stepvariation of salt concentration such as NaCl concentration or theconcentration of other inorganic salts (e.g., inorganic saltcombinations from the Hofmeister series of ions), Arginine and otheramino acids such as histidine, proline, phenylalanine, tyrosine,tryptophan, and glycine concentration, use of mild denaturing agentssuch as, for example, Polysorbate 20, Polysorbate 80, Polyethyleneglycol 2000, Polyethylene glycol 8000, Triton X-100, CHAPS, NP-40, andother ionic, non-ionic and/or zwitterionic surfactants, and so on.

LambdaFabSelect Chromatography Step:

An anti-IL-6Rc/IL-6RC κλ-body bispecific IgG antibody was purified usingLambdaFabSelect affinity chromatography media (GE Healthcare). The heavyand light chain amino acid sequences of the anti-IL-6Rc/IL-6RC κλ-bodybispecific IgG antibody are shown below:

Anti-IL6RC VKappa light chain (SEQ ID NO: 4)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWLPTTPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECAnti-IL6RC VLambda light chain (SEQ ID NO: 7)QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSWDAEFRAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECSCommon heavy chain (SEQ ID NO: 6)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

After column loading at 20 mg/mL and a wash step with 50 mM SodiumPhosphate, 250 mM Sodium Chloride, pH 7.0 (5 column volumes), a pHstep-elution was performed using a 50 mM glycine buffer adjusted at pH3.0. The flow through and eluted fractions were collected and analyzedby absorbance measurement at 280 nm (using a NanoDrop UV-Visspectrophotometer, Thermo Scientific) in order to determine productrecovery, reduced and non-reduced SDS-PAGE (using Invitrogen NovexNuPAGE 12-well 4-20% gradient gels following manufacturer's guidelines)in order to determine the purity and composition of the samples andhydrophobic-high performance liquid chromatography (HIC-HPLC; methoddescribed below) in order to determine the ability of the purificationprocess to separate the κλ-body bispecific IgG from the two monospecificantibody by-products.

HIC-HPLC Method:

In order to determine the relative proportions of the λ-MAb, κ-MAb andthe κλ-body in a sample mixture, a HIC-HPLC (hydrophobic interactionchromatography) assay using a Dionex ProPac HIC-10 column was used. Adescending gradient between 85 to 25% of ammonium sulfate was appliedonto the column after the loading of the sample in order to eluate the 3species with high resolution, the κ-MAb eluting first, followed by theκλ-body and finally the λ-MAb. Peak area integration of the UV tracemonitored at 280 nm was performed in order to determine the amount ofeach species.

As shown by the UV trace in FIG. 4A, the pH step elution applied to theLambdaFabSelect chromatography resin allowed the purification of theκλ-body. Non-reduced SDS-PAGE analysis, shown in FIG. 4B, revealed thehigh purity of the purified fraction containing assembled full lengthantibodies as anticipated. Some free light chain products (monomer anddimer forms) were also detected by non reduced SDS-PAGE. The purifiedfraction was further characterized by integrating the peak areas of theHIC-HPLC chromatograms (FIG. 4C). The results summarized in Table 2 werein accordance with the SDS-PAGE analysis, demonstrating the vastabundance of the κλ-body (89.4%) in the eluate fraction at pH 3.0.

TABLE 2 UV peak integration of HIC-HPLC analysis of LambdaFabSelectbound fractions % area Samples mono-λ κλ-body mono-κ LambdaFabSelect Notdetected Not detected 100.0% flow-through LambdaFabSelect  89.4% 10.6%Not detected pH 3.0 eluate LambdaFabSelect 100.0% Not detected Notdetected strip

Free Light Chain Reduction Using Mep HyperCel™ Mixed ModeChromatography:

To decrease manufacturing costs, the biotech/pharmaceutical industry isdeveloping purification processes that omit the initial proteinA-affinity chromatography step. Alternative purification solutions aretherefore currently being explored. In particular, mixed-modechromatography offers novel selectivity exploiting a combination of bothionic and hydrophobic interactions allowing for selective isolation ofantibodies from the cell culture contaminants. These contaminants caninclude host cell proteins, cellular DNA, endotoxins, viruses, as wellas antibody fragments. As described above, mammalian cells expressingrecombinant antibodies also secrete non-assembled free light chains intothe supernatant.

The present invention relates to the efficient removal of free lightchains from monospecific and bispecific antibodies. In particular,chromatography conditions have been identified that are applicable forbispecific or monospecific monoclonal antibodies and free light chains.The present invention is illustrated by a method of reducing free lightchain contaminants from the supernatant of a CHO cell line expressing aκλ-body (see FIG. 5A-5C). The method comprises the following steps: a)applying the cell culture supernatant to a solid chromatographymixed-mode resin (e.g., MEP HyperCel), b) eluting the monoclonalantibody with an acetate-buffered elution buffer at a pH 5.0 (eluate),and c) removing free light chains which are strongly bound to the resinat pH 2.1 (strip).

FIG. 5A shows a representative MEP HyperCel chromatogram demonstratingthe presence of FLC in the strip as determined by non-reduced SDS PAGE(FIG. 5B). SEC HPLC analysis confirmed efficient FLC removal from 60% inthe cell culture supernatant down to 33% in the antibody eluate fraction(FIG. 5C) and Table 3 below.

TABLE 3 Analysis of Mep HyperCel ™ chromatography fractions by SEC-HPLC.High molecular Free light weight species IgG monomer chains Unprocessed1.8% 32.2% 66.0% bulk harvest Flow through Not detected Not detected Notdetected Eluate 0.9% 66.1% 33.0% Strip Not detected Not detected 100.0% 

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1-8. (canceled)
 9. A method of purifying at least one bispecificantibody from a mixture, the method comprising the steps of: (a)providing a mixed antibody composition that includes one or morebispecific antibodies and at least one non-intact antibody molecule,where the non-intact antibody molecule comprises one or more antibodycomponents, one or more dimers of an antibody component, one or moreantibody fragments, and combinations thereof; (b) providing a separationmeans that has a differential affinity between a bispecific antibody anda non-intact antibody molecule; (c) contacting the separation means withthe mixed antibody composition under conditions that allow fordifferential binding to the separation means by the one or morebispecific antibodies as compared to the binding to the separation meansby the non-intact antibody molecule; and (d) separating the one or morebispecific antibodies from the separation means and retaining the one ormore bispecific antibodies.
 10. The method of claim 9, wherein thenon-intact antibody molecule is a free light chain.
 11. The method ofclaim 9, wherein the separation means is a resin, a membrane, a magneticbead, a particle or a monolith.
 12. The method of claim 9, wherein theseparation means is coupled to a ligand having high specificity andaffinity for a bispecific antibody molecule.
 13. The method of claim 12,wherein the separation means is a mixed mode chromatography resin. 14.The method of claim 13, wherein the separation means is a Mep HyperCel™resin.
 15. The method of claim 9, wherein step (d) comprises eluting thebispecific antibody or combination of bispecific antibodies from theseparation means under conditions that allow for preferential detachmentof the at least one bispecific antibodies from the separation means ascompared to the detachment of the non-intact antibody molecule.
 16. Themethod of claim 9, wherein step (c) comprises contacting the separationmeans under conditions that allow for the binding of the one or morebispecific antibodies to the separation means but do not allow for thebinding of the non-intact antibody molecule to the separation means. 17.The method of claim 16, wherein step (d) comprises eluting thebispecific antibody or combination of bispecific antibodies from theseparation means under conditions that allow for detachment of the atleast one bispecific antibody.
 18. The method of claim 16, wherein step(c) further comprises the step of removing any unbound, non-intactantibody molecules from the composition.
 19. A method of purifying atleast one bispecific antibody from a mixture, the method comprising thesteps of: (a) providing a mixed antibody composition that includes oneor more bispecific antibodies and at least one non-intact antibodymolecule, where the non-intact antibody molecule comprises one or moreantibody components, one or more dimers of an antibody component, one ormore antibody fragments, and combinations thereof; (b) providing aseparation means that has a differential affinity between an intactantibody and a non-intact antibody molecule; (c) contacting theseparation means with the mixed antibody composition under conditionsthat allow for binding to the separation means by the non-intactantibody molecule but do not allow for binding to the separation meansby the one or more bispecific antibodies such that the one or morebispecific antibodies remain in an unbound fraction; and (d) retainingthe unbound fraction comprising one or more bispecific antibodies. 20.The method of claim 19, wherein the non-intact antibody molecule is afree light chain.
 21. The method of claim 19, wherein the separationmeans is a resin, a membrane, a magnetic bead, a particle or a monolith.22. The method of claim 19, wherein the separation means is coupled to aligand having high specificity and affinity for a non-intact antibodymolecule.
 23. The method of claim 22, wherein the separation means is amixed mode chromatography resin.
 24. The method of claim 23, wherein theseparation means is a Mep HyperCel™ resin.
 25. The method of claim 15,wherein the step of eluting comprises an acetate elution buffer at pH5.0.